Optical spectroscopic study of the effects of a single deoxyribose substitution in a ribose backbone: implications in RNA-RNA interaction.

The 2'-OH group in the ribose sugars of an RNA molecule plays an important role in guiding tertiary interactions that stabilize different RNA structural motifs. Deoxyribose, or 2'-OH by 2'-H, substitution in both the single-stranded and the duplex part of an RNA backbone has been routinely used to evaluate what role the 2'-OH plays in different tertiary interactions that guide an RNA-RNA contact. A deoxyribose substitution not only has the effect of removing a hydrogen bond donating group, but also introduces a sugar moiety with a preference for C2'-endo pucker in a backbone of predominantly C3'-endo sugars. This study evaluates the effects of a single deoxyribose substitution in both single-stranded and double-helical forms of RNA oligomers. A single-stranded, nonrepetitive 7-mer oligoribonucleotide (7-mer RNA) and four different variants having the same base sequence but with a single deoxyribose sugar at different positions in the strands have been studied by ultraviolet (UV) absorption, circular dichroism (CD), and Fourier transform infrared (FTIR) spectroscopy. Duplexes were formed by association with the complementary strand of the 7-mer RNA. The results show that both RNA and DNA single strands have preorganized conformations with spectral properties resembling those of A- and B-form helices, respectively, with RNA being more heterogeneous than its DNA counterpart. A single deoxyribose substitution perturbs the structure of the RNA backbone, with the effect being more pronounced in the single-stranded than in the duplex structure. The perturbation depends on the position of the 2'-H substitution in the strand.

FTIR spectroscopic studies of oligonucleotides that model a triple-helical domain in self-splicing group I introns.

Fourier Transform infrared (FTIR) spectroscopy was used to characterize the Mg2+ dependent association of a 23-mer mixed ribo-deoxyribonucleotide (23-mer RNA) and a 7-mer oligoribonucleotide (7-mer RNA) that models the triple-helical domain of a self-splicing group I intron [Sarkar et al. (1996) Biochemistry 35, 4678-4688]. To elucidate the effect of deoxyribose substitution in the entire backbone, as well as at specific positions, in the assembly of the triple-helical domain, parallel studies were carried out on the association of pure deoxyribonucleotides having base sequences corresponding to the oligoribonucleotides and also between 23-mer RNA and two 7-mer RNA variants. In the variants, either the ribose attached to G451 or the ribose attached to U453 was changed to deoxyribose. FTIR-monitored thermal denaturation of the two 23-mer hairpins shows two distinct melting regions in 1 M NaCl, in case of the RNA hairpin but not for the 23-mer DNA. Triple-helix association between the two strands (7-mer and 23-mer) studied by FTIR show that only when both strands are RNA, association takes place with the formation of the P6 helix. Our results also show that the interactions between the two RNA strands involve some participation of the riboses, which could also involve the 2'-OH groups of the RNA backbone. The assembly of the triple-helical domain is not possible with a deoxyribose backbone and is completely perturbed even when only one ribose at either G451 or U453 position is substituted by deoxyribose.

A synthetic model for triple-helical domains in self-splicing group I introns studied by ultraviolet and circular dichroism spectroscopy.

Structural studies were performed on synthetic oligonucleotides with sequences corresponding to the P4/P6 and J3/4, J6/7 regions of the self-splicing group I intron of the bacteriophage T4 nrdB pre-mRNA, which correspond to the proposed triple-helical domain in the Tetrahymena thermophila intron. A 23-mer RNA was synthesized as a mixed ribo-deoxyribo oligonucleotide, modeling an expected base-paired region of P4 along with the J3/4 and P6 (5'-end bases of P6) regions. strand modeling the 3'-end bases of P6 and J6/7 regions, with which a triple helix may form, was synthesized as a pure oligoribonucleotide (7-mer RNA). The interactions of these oligonucleotides have been characterized by UV and circular dichroism (CD) spectroscopy. The results show that the 23-mer RNA forms a stable hairpin modeling the P4 base-paired region. Triple helix association between the 23-mer RNA hairpin and the 7-mer RNA single strand was detected by CD in the presence of Mg2+ (>5mM) but not in presence of a monovalent cation like Na+ (up to 500 mM). Studies on selected variants of both 7-mer and 23-mer RNAs were carried out. The results show that for the association of the two partner strands not only the formation of P6 helix but also triplet interactions between two strands are required. The association of the two strands in general follow a pattern predicted by comparative sequence analysis. Parallel studies on pure oligoribonucleotides having base sequence corresponding to those of the oligoribonucleotides showed no evidence of association under similar conditions, which could indicate that the 2'-hydroxyl groups of the riboses might play an important role in hydrogen bonding to form the required nucleoside triples. Molecular modeling studies on the proposed "plaited triple helix" formed by the association of the 23-mer RNA hairpin and 7-mer RNA single strand showed that the structure is sterically and energetically feasible.

Evaluation of 2'-hydroxyl protection in RNA-synthesis using the H-phosphonate approach.

A number of different protecting groups were compared with respect to their usefulness for protection of 2'-hydroxyl functions during synthesis of oligoribonucleotides using the H-phosphonate approach. The comparison was between the t-butyldimethylsilyl (t-BDMSi), the o-chlorobenzoyl (o-CIBz), the tetrahydropyranyl (THP), the 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp), the 1-(2-chloro-4-methylphenyl)-4-methoxypiperidin-4-yl (Ctmp), and the 1-(2-chloroethoxy)ethyl (Cee) protecting groups. All these groups were tested in synthesis of dodecamers, (Up)11U and (Up)11A, using 5'-O-(4-monomethoxytrityl) or (4,4'-dimethoxytrityl) uridine H-phosphonate building blocks carrying the respective 2'-protection. The performance of the t-BDMSi and o-CIBz derivatives were also compared in synthesis of (Up)19U. The most successful syntheses were clearly those where the t-butyldimethylsilyl group was used. The o-chlorobenzoyl group also gave satisfactory results but seems somewhat limited with respect to synthesis of longer oligomers. The results with all tested acetal derivatives (Fpmp, Ctmp, Cee, THP) were much less successful due to some accompanying cleavage of internucleotidic H-phosphonate functions during removal of 5'-O-protection (DMT).

[Synthesis of oligoribonucleotides by the N-phosphonate method using alkali-labile 2'-o-protective groups. II. Various aspects of using 2'-o-benzoyl and anisole protective groups]. / Sintez oligoribonukleotidov N-fosfonatnym metodom s izpol'zovaniem shchelochnolabil'nykh 2'-o-zashchitnykh grupp. II. Nekotorye aspekty izpol'zovaniia 2'-o-benzoil'noi i anizoilnoi zashchitnykh grupp.

The N-acyl, 5'-O-trityl (MeOTr, (MeO)2Tr, Me3Tr), 2'-O-benzoyl (and anisole) nucleosides were prepared by selective aroylation of N,5'-protected nucleosides. By means of the reverse-phase microcolumn liquid chromatography it was shown that the rate of the aryl 2'----3'-isomerisation is lower in case of 2'-anisoylnucleosides and depends on structure of the 5'-O-protecting group. The prepared synthons were used for the manual H-phosphonate solid-phase synthesis of oligoribonucleotides (6-10-mers).